Multi-well plates are used to perform various biological, chemical or biochemical assays. A multi-well well plate is composed of a two-dimensional array (e.g., 12×8) frustoconical wells extending into, and typically through, a planar plate from a major surface of the plate. Liquid specimens such as blood, plasma, serum, urine and various reagents are placed into the wells. In some instances, it is desired to monitor the progress of the reactions taking place in the wells. One way to do this is to attach a fluorescent dye to one of the participants in the reaction. The wells are irradiated with excitation light of a wavelength that stimulates the fluorescent dye and the intensity of resulting emission light is measured.
One type of analysis performed in multi-well well plate is a real-time polymerase chain reaction (qPCR) that may be used in DNA sequencing, DNA cloning, gene mapping, and other forms of nucleic acid sequence analysis. In general, qPCR relies on the ability of DNA-copying enzymes to remain stable at high temperatures. A specimen containing DNA molecules is placed in one or more wells of the well plate together with various reagents including a DNA-binding fluorescent dye. The well plate is heated to over 90° C. to break the bonds between the two strands that constitute the DNA molecules in the specimen. The well plate is next cooled to about 0° C. At this temperature, primers bind to the ends of the strands. Finally, the well plate is heated to about 75° C. At this temperature, nucleotides add to the primers and eventually a complementary copy of the DNA template is formed. Binding to the DNA molecule activates the fluorescent dye. Consequently, the intensity of the emission light output by the activated fluorescent dye provides a measure of the amount of the fluorescent dye that has been activated, and, hence, the number of DNA molecules that have been produced.
Conventional instruments for measuring the intensity of the emission light generated by the activated fluorescent dye in qPCR and other reactions monitored by fluorescent dyes use such devices as cameras, photodiodes and photomultipliers to detect the light emitted by the fluorescent dye. Such conventional instruments are configured to work with a specific fluorescent dye, i.e., the instrument generates excitation light at the specified excitation wavelength of the specified fluorescent dye and includes narrow-band emission light filters at the specified emission wavelength of the specified fluorescent dye. This limits the instrument to use with the specified dye. The instrument cannot be used with newer, better, fluorescent dyes that are later introduced. Moreover, different fluorescent dyes cannot be used in different wells of the multi-well plate in a camera-based instrument because all the emission light passes through a single emission filter. Photodiode- and photomultiplier-based instruments are also limited to specific fluorescent dyes, and additionally cannot make simultaneous measurements in multiple wells because such instruments use an X-Y mechanical scanner to align the optical system with the wells of the well plate sequentially.
Additionally, conventional instruments for measuring the intensity of the emission light generated by a fluorescent dye require regular calibration. A conventional instrument has to be taken off-line for calibration and is not available to be used to perform measurements while it is being calibrated.
Accordingly, what is needed is an instrument for measuring the intensity of the emission light generated by an activated fluorescent dye during, for example, qPCR that can be used with multiple different fluorescent dyes, including fluorescent dyes that are not commercially available at the time the instrument is manufactured, that can measure the intensity of the fluorescent light in multiple wells simultaneously, that can measure the intensity of the fluorescent light generated by different fluorescent dyes sequentially, and that does not require the instrument to be taken off-line for calibration.